Hydrogen bonds yellow stabilize the antibody-antigen interaction. In addition to hydrogen bonds, other weak interactions such as van der Waals forces, hydrophobic interactions and electrostatic forces improve the binding specificity between antibody and antigen. These interactions occur over large and sometimes discontinuous regions of the molecules, improving binding affinity. The animation shows amino acids of the antibody that interact with Gln Animation: Gln of lysozyme surrounded by amino acid residues of antibody.
Molecular structures represented in this tutorial were obtained by X-ray crystallography. Antibody Structure Introduction. Water molecules light blue fill in spaces between the antigen and the antibody.
The water molecules contribute significantly to the binding energy by creating additional hydrogen bonds. Consequently, they hypothesized that conformational changes in Fab molecules were occurring upon Ag binding, and were most likely being transferred through the elbow angle. Furthermore, Pritsch et al. Their model also showed that this loop had a different conformation for each isotype Pritsch et al.
This work supports the hypothesis that the C H1 domain has an allosteric role in Ag binding and may also be transmitting structural signals to the rest of the Ig as suggested by Huber et al. McLean et al. Furthermore, they saw differences between the various chimeric Abs upon binding monovalent and multivalent Ags, as well as differences in their binding location to a microbial capsule McLean et al.
Further SPR studies using this family of mAbs compared the intact murine IgG 1 with its chimeric form as well as its deglycosylated form. These studies revealed that glycosylation did not alter Ag binding kinetics and there were no significant differences in Ag binding between the original mAb and its murine—human chimeric form Torres et al.
However, it is unclear whether C region glycosylation may play a role in the Ag binding of other families of isotypes, as this has not been thoroughly studied. A series of studies with the 3E5 family of murine isotypes to Cryptococcus neoformans polysaccharide also identified significant changes in isotype specificities and affinities.
Among the isotypes, the IgG 1 Fab showed the most favorable binding parameters. This implied that differences in specificity among the isotypes were potentially due to differences in the C H1 region alone Torres et al. ITC studies done with the 3E5 family using full IgG molecules and the P1 peptide Ag, confirmed a binding stoichiometry of peptide:Ab as well as significantly different association constants between all four isotypes Dam et al. In , Tudor et al.
They also found altered epitope specificity and increases in anti-HIV-1 activity assays, indicating significant changes to the Ig paratope Tudor et al. A study by Crespillo et al. Their results showed significant differences in binding affinities between the different forms of Ab and monomeric peptide Ag epitopes, with highest affinities achieved with whole IgG Crespillo et al.
More recently, Xia et al. They also studied Trp fluorescence and circular dichroism with these isotypes and observed changes upon Ag binding that were isotype dependent Xia et al. For a set of Abs binding to the Bacillus anthracis capsule class-switching from the original IgG 3 to IgG 1 , IgG 2a , and IgG 2b isotypes resulted in a loss of protection, affinity and a change in mAb binding to its capsular Ag Hovenden et al.
Hovenden et al. This contrasts with earlier studies that reported similar binding differences between Fab fragments and whole IgG, suggesting an allosteric role for the C H1 domain Yuan et al. Furthermore, Hovenden et al. Although they could not exclude a contribution from Fc—Fc interactions, there was evidence that other factors must also be contributing to the observed changes in affinity Hovenden et al.
Using their family of isotypes, they were also able to exclude the hypothesis that as flexibility of the hinge region increases, Ag binding affinity increases.
This hypothesis had been suggested by Morelock et al. These studies shed light on the different behaviors of different families of mAb isotypes and the need to further investigate and understand how C regions affect paratope properties and how this may differ between different mAb families.
Recently, several studies have identified additional instances where Abs with identical V regions differing in isotype manifest differences in Ag binding. In fact, they found that the IgA 2 bound one Ag strain with 2. Another group studying the Phl p7 grass pollen allergen generated a full panel of human IgG and IgA isotypes and found subtle but significant differences in binding rates using SPR.
They observed the greatest differences in both on- and off-rate constants to be about three-fold, which canceled out to give an overall affinity range of — picomoles pM Dodev et al.
In addition to the various examples of V region-identical Igs exhibiting specificity differences, it is important to note that several studies have reported V region-identical Igs with no changes in specificity. Although the failure to detect changes could be the result of insufficient sensitivity in the assays done, it is possible that the phenomenon of C-mediated changes in specificity and affinity is associated with some V regions and not others.
Consequently we compared unique V H and V L sequences from 24 Igs with V region-identical isotype switch variants compiled from the literature Table 2. An analysis of the sequence similarities and germline gene hits for these Igs shows that these appear to group phylogenetically, consistently with the notion that certain V region gene families may differ in being permissive or non-permissive of specificity changes following class switching Figure 2.
It is interesting to note that all of the human lambda V L Igs were non-permissive. With the small number of Ig sequences available for this analysis, no firm conclusions can be made and more examples will be needed to confirm a V region germline basis for C domain-mediated specificity changes.
It will also be useful to compare the structures of Igs with very similar V regions that differ in specificity to determine whether they are permissive of specificity changes, such as V L sequences 6, 8, and 11 in Table 2. Furthermore, the combination of specific V H and V L genes is another potential variable for C domain-mediated specificity changes since it is conceivable that even for permissive V regions that expression of this effect requires combination with certain C regions.
In this regard it is noteworthy that different C regions combined with the same V region manifested differences in the magnitude of the changes observed Janda et al. Table 2. Unique antibodies with variable-region-identical isotype-switch variants and identifiable amino acid sequences.
Figure 2. Relationships between permissive and non-permissive V region sequences. Immunoglobulins with differing C regions and identical V regions were identified in the literature Table 2. VH and VL amino acid sequences were found for 24 of these unique antibodies 11 human and 13 murine, Table 2. For each group, a dendrogram was constructed through hierarchical average-linkage clustering with pairwise sequence similarity calculated as the Levenshtein distance.
Leaf labels in the dendrograms are colored according to whether changes in the constant region for that antibody were permissive green or non-permissive red of specificity changes. These studies necessitate expanding the role for isotype class switching to a new role where it contributes to the generation of Ig diversity. A recent review by Sela-Culang et al. They suggested that this could re-shape the Ag binding site and as such could be considered as a mechanism for generating Ab diversity Sela-Culang et al.
This is further supported by the hypothesis that the conformational diversity of Abs is directly linked to Ab multispecificity and supports the role of a single sequence i. Since the mechanisms of isotype switching and somatic mutation share some of the same proteins this notion has the elegance of bringing together these two processes within the same molecular pathways. Isotype class switching has further implications for primary and secondary B cell responses, idiotype reactivity and immunogenicity.
Also, the observation that Ig class switching can result in reactivity for self Ags despite identical V regions suggests that this phenomenon may be implicated in the appearance of certain pathological autoimmune responses Torres et al. An understanding of how C regions affect Ab paratope is important for the development of therapeutic mAbs Nosanchuk, A plausible mechanism on the molecular level for the first of these effects involves C-mediated structural constraints on V region structure that affect the conformation of the Ig paratope.
In contrast, C region glycosylation has thus far not been shown to contribute to this phenomenon Torres et al. In , Greenspan et al. The Fc regions were found to be required for this enhanced binding. Subsequently, Cooper et al. Cooper et al. While the IgG 1 and IgG 2b Abs and the IgG 3 F ab' 2 fragments bound best to the streptococcal strain with the highest epitope density, the IgG 3 Ab bound best to the strain with intermediate epitope density.
Additional experiments verified that despite these differences in binding to multivalent Ags, the three Abs IgG 3 , IgG 1 , and IgG 2b bound to monovalent Ag or multiple rat anti-idiotypic mAbs comparably Cooper et al. Thus, in spite of identical V domain amino acid sequences and ability to bind monovalent hapten, the IgG 3 Ab discriminated among multivalent antigens differently than the IgG 1 or IgG 2b Abs i.
Additional conclusions drawn by the authors are that epitope density and H chain C region structural differences can contribute to differences in multivalent binding among IgG subclasses. Analysis of this Ab-Ag system with SPR revealed that the stronger binding of the cooperative IgG 3 Ab, in comparison to the non-cooperative IgG 1 and IgG 2b Abs, was associated with both greater on rates and slower off rates, consistent with the hypothesis that non-covalent Fc—Fc interactions mediated the cooperativity of the IgG 3 Ab Cooper et al.
Also in , Schreiber et al. In roughly the same time period, Izui and colleagues published a number of papers focused on murine IgG 3 cryoglobulins in autoimmune disease models that reported data consistent with the results of Greenspan and his associates. For example, Fulpius et al. Another group with similar results on isotype differences in avidity during this time used a set of chimeric mAbs murine V region, human C region against both monovalent and bivalent intercellular adhesion molecule 1.
Though full-length mAbs showed differences in competition ELISA, their murine and chimeric Fab counterparts had equivalent binding constants, indicating that any differences in whole mAb were due to differences in avidity, and not monovalent affinity Morelock et al. Circular dichroism studies done on a family of murine IgGs to C. This was followed by tryptophan fluorescence studies of the same family of murine IgG Abs which showed different changes in electrical properties of Ig Fab Trp molecules some of which are in the paratope, upon Ag binding.
NMR studies of the same group of Igs further expanded the notion by showing differences in the chemical environments of their paratopes, as well as IgE and IgA isotypes. Finally, X-ray crystallographic studies and molecular modeling of these Igs identified structural differences that occur mainly in the hinge angles among Fab molecules of the IgG 1 and IgG 3 isotypes Janda et al. In Adachi et al. In addition, Adachi et al. The difference in water molecules in the interface may represent a much tighter interaction for the Fab-HEL complex, which may be essential for HEL binding and could explain the differences in dissociation factors.
The authors hypothesized that removal of the C domains in the Fv molecule may result in imperfect complementarity between Ab and Ag, and thus lead to an increase in water molecules between paratope and epitope Adachi et al. Furthermore, the UL2-C L region, which is highly conserved in human and murine light chains, was previously predicted to have unique fluctuations corresponding with Ag binding Kabat et al. These new studies indicate that the UL2—C L fluctuations may be playing an important role in allosteric mediation of paratope—epitope interactions Adachi et al.
In , Xia et al. In addition, they found significant differences in histone and kidney Ag binding profiles using SPR, and different changes in Trp fluorescence upon Ag binding.
This parameter can therefore have a strong impact on the binding affinity. Indeed, the binding energy between two atoms is a function of their distance following the Lennard-Jones relation.
A difference of a few Angstroms can strongly affect the value of the binding free energy. The choice of the framework regions for humanization by the CDR-grafting technique is therefore of crucial importance to maintain affinity. For example, Nakanishi et al. Similarly, Bujotzek et al. Framework residues affecting significantly the affinity are listed in Table S2.
To achieve a high affinity binding, the paratope and its corresponding epitope must have large shape complementary surfaces and, in addition, the contacting residues must establish interactions that stabilize the complex. The parameters that influence the shape diversity of the paratope are essentially the CDR lengths and conformations canonical classes 32 , 56 , 64 , 81 , their relative orientations 77 — 79 , 82 and the hydration shell solvation of the binding interface The binding of the two complementary surfaces is mostly driven by aromatic residues that establish van der Waals and hydrophobic interactions while the strengthening of the complex involves rather electrostatic interactions and hydrogen bonds established between side chains of adequately positioned charged and polar residues.
An antibody humanization experiment attempts to reconstitute the original paratope-epitope interactions, in most cases, by grafting the CDRs of a non-human antibody to a human antibody scaffold. This CDR-grafting or reshaping method is often based on a simplified view of antigen-antibody interaction that reduces the paratope to the 6 CDRs of the antibody.
Although, there is no general protocol to perform an antibody humanization, since it is always a case-to-case experiment, this section attempts to provide guidance in such an exercise.
Figure 8 summarizes and suggests a standardized protocol to humanize antibodies from animal origins. Firstly, we need to identify the CDRs within the loop of the donor antibody from animal origin.
Under the simplified assumption that the paratope corresponds to the CDRs, it is recommended using the Chothia's CDR definition as they correlate very well with the structural loops present in the variable regions. A few online software tools are available for CDR structure prediction 10 , 11 72 , 84 and classification 12 However, it is always best to choose the broadest CDR definition to ensure that all residues constituting the paratope will be included.
Secondly, as discussed repeatedly in this review, residues outside the classical CDR definitions can also be part of the actual paratope. Different studies can help to identify these residues, but unfortunately, only one bioinformatical tool Paratome is currently available.
Finally, the relative orientation of the CDRs is also critical to reconstruct the paratope surface and to position adequately its antigen interacting residues.
Hence, it is essential to choose the most appropriate human V H and V L framework scaffolds. Selecting this human antibody scaffold is probably as important as the definition of the CDRs.
Figure 8. Representation of a standardized humanization protocol. Prediction tools are indicated in blue. Noticeably, for all these purposes, it is important to align the antibody sequences correctly and to identify precisely the residues with superimposed positions in chains of different origins. Therefore, the handling of the same and an adequate numbering scheme that attributes an identical number of residues including fixed possible residue insertion points to occupy the same structural positions in the immunoglobulin chains forms a prerequisite for all antibody engineering tasks.
In this respect, the enhanced Chothia's Martin's numbering system is a bit easier to use since it identifies precisely insertion points but, of course, this choice is quite subjective. Another critical point is that the humanized antibodies should not induce any adverse immune reaction in the patient.
Therefore, prediction of potential immunogenic epitopes in the protein sequence should be performed. Briefly, this adverse immune response takes place after internalization of the antibodies by the antigen presenting cells. These membrane protein complexes bind specific oligopeptides and present them to lymphocytes T helpers that activate the immune response.
Nowadays, databanks reporting the polymorphisms of these MHC-II molecule alleles are available to predict the oligopeptide binding potential 85 — One of these is the Epivax web tool 88 that consists in scanning a protein sequence for identifying putative T cell epitopes. This software offers to mutate one or more amino acids in order to reduce the immunogenicity of a protein sequence. It is important to realize, that in the case of antibodies, conflicts may arise between maintaining a high affinity and a low immunogenicity.
This is one of the reasons why antibody humanization remains challenging. This review describes the different amino acid numbering systems and CDR definitions that are currently available and it highlights the importance of standardized numbering system for antibody engineering strategies, especially for antibody humanization tasks.
Indeed, an effective amino acid numbering system should be able to assign the same number of residues to structurally aligned positions in antibodies from different species. Although several numbering tools based on ever growing databases are available online, it is recommended to compare the different numbering systems as inaccuracies are still possible, especially for variable antibody domains with unconventional lengths. Furthermore, the different CDR definitions and other concepts, such as contact residues or antigen binding residues, have been reviewed.
In the context of antibody humanization methods, paratope has been very often limited to the CDRs. This approximation is useful as long as it permits the CDR-grafting method to be an easy and generalizable tool for humanization. However, this approach is less convenient in antibody humanization efforts because of the discontinuous nature of the paratope combined with the experimental approaches that require the precise determination of these residues.
Moreover, different studies that have analyzed the angle between the light and heavy chain variable regions have been described. Finally, all of these concepts that are crucial for the humanization of antibodies should be included in the humanization process. In summary, a precise identification of the paratope established using an appropriate amino acid numbering scheme is necessary to engineer humanized antibodies with high affinity, stability and low immunogenicity.
All these residues have to occupy identical positions in the 3D structure. MD and MV wrote the manuscript. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Table S1. VL states for variable domains of light chains, VH for variable domains of heavy chains. IMGT and Honegger Aho numbering systems do not take into account the nature heavy or light of the chain. Insertion positions are highlighted in yellow. In the Aho numbering scheme, these yellow residues correspond to the gap positions. Deletion positions are in brackets residue L 10 in lambda light chains, Martin numbering scheme.
The Gelfand numbering scheme is divided in words boxes with the numbering below. For the Aho numbering scheme, structurally conserved position are highlighted in green.
Table S2. CDR definitions are indicated in orange. The upper case letter refers to the publication that describes the impact of the residue. Clin Transplant. Smith S. J Transpl Coord — Feldmann M, Maini RN. Annu rev Immunol. Structural consensus among antibodies defines the antigen binding site.
PLoS Comput Biol. Restricted diversity of antigen binding residues of antibodies revealed by computational alanine scanning of antibody—antigen complexes.
J Mol Biol. Antibody-antigen interactions: contact analysis and binding site topography. Comparative analysis of nanobody sequence and structure data. Proteins Struct Funct Bioinforma — Nelson AL. Antibody fragments: Hope and hype. MAbs — The coming of age of engineered multivalent antibodies.
Drug Discov Today — Vigne E, Sassoon I. Adverse events of monoclonal antibodies used for cancer therapy. Biomed Res Int. Not four letter words. Google Scholar. Human antiglobulin response to foreign antibodies: therapeutic benefit?
Cancer Immunol Immunother. J Immunol — Replacing the complementarity-determining regions in a human antibody with those from a mouse. Nature — Comparison of surface accessible residues in human and murine immunoglobulin Fv domains. Implication for humanization of murine antibodies. Padlan EA. A possible procedure for reducing the immunogenicity of antibody variable domains while preserving their ligand-binding properties.
Mol Immunol. PubMed Abstract Google Scholar. J Immunol. A molecular immunology approach to antibody humanization and functional optimization.
Guiding the selection of human antibodies from phage display repertoires to a single epitope of an antigen. Biotechnology — The fragment antigen-binding Fab fragment is a region on an antibody that binds to antigens.
It is composed of one constant and one variable domain of each of the heavy and the light chain. The two variable domains bind the epitope on their specific antigens. In an experimental setting, Fc and Fab fragments can be generated in the laboratory.
The enzyme papain can be used to cleave an immunoglobulin monomer into two Fab fragments and an Fc fragment. The enzyme pepsin cleaves below hinge region, so a F ab' 2 fragment and a pFc' fragment is formed. The F ab' 2 fragment can be split into two Fab' fragments by mild reduction. The variable regions of the heavy and light chains can be fused together to form a single-chain variable fragment scFv , which is only half the size of the Fab fragment, yet retains the original specificity of the parent immunoglobulin.
A diabody is two scFvs with connected with linker peptides that are too short for the two variable regions to fold together about five amino acids , forcing the scFvs to dimerize.
Diabodies have been shown to have dissociation constants up to fold lower than corresponding scFvs, meaning that they have a much higher affinity to their target. Two scFv's which bind to the same or different antigens may also be connected with longer linkers such as leucine zippers. Educational Appendix.
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